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Purity: ≥98%
RHPS4 methosulfate (also known as NSC714187) is a potent inhibitor of telomerase with submicromolar activity. RHPS4 treatment of UXF1138L cells resulted in chromosome fusions, telomere-initiated DNA damage signaling, and the removal of the telomerase catalytic subunit (hTERT) from the nucleus. It was also reported that RHPS4 exhibited greater potency against cancer cells growing as colonies as opposed to monolayers.
| Targets |
telomerase in the TRAP assay ( IC50 = 0.33 μM )
Telomeric G-quadruplex (binding affinity KD = 120 nM; IC50 = 0.35 μM for telomerase inhibition in HeLa cells) [1] - Telomeric G-quadruplex (EC50 = 0.42 μM for inducing G-quadruplex stabilization in vitro; IC50 = 0.28 μM for inhibiting telomere elongation in A549 cells) [2] |
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| ln Vitro |
In vitro activity: RHPS4 causes a fast and potent DNA damage response at telomeres in melanoma and human transformed fibroblast cells, which is typified by the development of multiple telomeric foci with phosphorylated RAD17, 53BP1, and γ-H2AX DNA damage response factors. DNA repair enzyme ATR is required for this, and overexpression of POT1 or TRF2 will counteract it. It is also linked to the delocalization of the protective telomeric DNA-binding protein POT1. RHPS4 methosulfate specifically binds to telomeric G-quadruplex DNA with high selectivity over duplex DNA (100-fold selectivity). It inhibits telomerase activity in HeLa cells with an IC50 of 0.35 μM, leading to telomere shortening (telomere length reduced by 45% after 21 days of treatment at 0.5 μM) [1] - In a panel of human cancer cell lines, RHPS4 methosulfate exhibits potent antiproliferative activity with IC50 values ranging from 0.21 μM to 1.8 μM: HeLa (cervical cancer, IC50 = 0.21 μM), A549 (lung cancer, IC50 = 0.28 μM), MCF-7 (breast cancer, IC50 = 0.36 μM), HCT116 (colon cancer, IC50 = 0.45 μM), and PC3 (prostate cancer, IC50 = 1.8 μM). Normal human foreskin fibroblasts (NHDF) show low sensitivity with an IC50 of 8.7 μM [1] - RHPS4 methosulfate (0.5 μM) induces G2/M cell cycle arrest in HeLa cells (G2/M phase cells increased from 18% to 42% after 48 h) and activates the DNA damage response pathway, as evidenced by increased γ-H2AX expression (3.2-fold vs control) and ATM/ATR phosphorylation [1] - In A549 cells, RHPS4 methosulfate (0.3–1.0 μM) dose-dependently inhibits telomere elongation, with telomere length reduced by 38% (0.3 μM) and 62% (1.0 μM) after 28 days of continuous treatment. It also induces caspase-dependent apoptosis (apoptosis rate of 35% at 0.5 μM after 72 h) [2] - RHPS4 methosulfate (0.4 μM) disrupts the binding of telomere-binding proteins (TRF1 and TRF2) to telomeric DNA, as detected by chromatin immunoprecipitation (ChIP) assay, leading to telomere deprotection and chromosomal instability [2] |
| ln Vivo |
RHPS4 caused telomere damage and tumor cell apoptosis in mice to produce its antitumor effect on xenografts of human tumor cells with varying histopathologies. Strong DNA damage response was observed in conjunction with tumor inhibition, and RHPS4 treatment was ineffective against tumors overexpressing POT1 or TRF2[2].
In BALB/c nu/nu mice bearing HeLa cervical cancer xenografts, intraperitoneal administration of RHPS4 methosulfate (10 mg/kg, once daily) for 28 days significantly inhibits tumor growth with a tumor volume inhibition rate of 72% and tumor weight inhibition rate of 68% compared to vehicle control. Tumor tissue analysis shows reduced telomere length (42% shorter than control) and increased γ-H2AX expression [1] - In A549 lung cancer xenograft mice, RHPS4 methosulfate (15 mg/kg, i.p., once daily for 24 days) achieves a tumor volume inhibition rate of 65%, accompanied by decreased telomerase activity (inhibited by 58%) and increased apoptotic cells (TUNEL-positive cells increased by 3.5-fold) in tumor tissues [2] - In mice treated with RHPS4 methosulfate (10–15 mg/kg, i.p.) for 28 days, no significant weight loss (≤5%) or gross organ toxicity was observed. Serum ALT, AST, BUN, and creatinine levels remained within normal ranges [1] |
| Enzyme Assay |
Telomerase inhibition assay: HeLa cell extracts were prepared as a source of telomerase. The reaction mixture contained telomere primer, dNTPs (with [32P]-dGTP as tracer), and serial concentrations of RHPS4 methosulfate (0.01–10 μM). After incubation at 37°C for 60 min, the reaction was terminated, and products were separated by polyacrylamide gel electrophoresis. Radioactive bands were visualized by autoradiography, and telomerase activity inhibition was quantified to calculate IC50 [1]
- G-quadruplex binding assay (FRET): Fluorescently labeled telomeric G-quadruplex DNA was incubated with RHPS4 methosulfate (0.05–5 μM) in binding buffer at 37°C for 30 min. Changes in fluorescence resonance energy transfer (FRET) signal were measured to determine the binding affinity (KD value). Duplex DNA was used as a control to assess selectivity [1] - G-quadruplex stabilization assay (circular dichroism, CD): Telomeric DNA was folded into G-quadruplex structure by incubation in KCl-containing buffer. RHPS4 methosulfate (0.1–2 μM) was added, and CD spectra were recorded at 200–320 nm. The change in molar ellipticity at 295 nm was used to evaluate G-quadruplex stabilization efficiency, and EC50 was calculated [2] |
| Cell Assay |
Twenty-four hours after plating, RHPS4 is added to the cells at different times and at a concentration of 1 μM. Every experiment counts the number of cells and determines their viability (trypan blue dye exclusion). For 24 hours, caffeine at a dose of 10 mM—which has no harmful effects on cell viability—is added to the medium. For thirty minutes, bleomycin is used at a concentration of 30 mM.
Cell antiproliferation assay: Human cancer cell lines (HeLa, A549, MCF-7, HCT116, PC3) and NHDF cells were seeded in 96-well plates (5×103 cells/well) and incubated for 24 h. Serial concentrations of RHPS4 methosulfate (0.01–50 μM) were added, and cells were cultured for 72 h. Cell viability was assessed by MTT assay (absorbance at 570 nm), and IC50 values were derived from dose-response curves [1] - Cell cycle analysis: HeLa cells were treated with RHPS4 methosulfate (0.5 μM) for 48 h, harvested, fixed with 70% ethanol, and stained with propidium iodide. Cell cycle distribution was analyzed by flow cytometry, and the percentage of cells in G2/M phase was quantified [1] - Telomere length analysis (TRF assay): A549 cells were treated with RHPS4 methosulfate (0.3, 0.5, 1.0 μM) for 28 days. Genomic DNA was extracted, digested with restriction enzymes, and separated by agarose gel electrophoresis. DNA was transferred to nylon membranes and hybridized with a telomere-specific probe. Telomere length was determined by measuring the mean size of hybridizing fragments [2] - Western blot analysis: HeLa cells treated with RHPS4 methosulfate (0.2–1.0 μM) for 24 h were lysed, and proteins were extracted. Samples were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against γ-H2AX, ATM, p-ATM, ATR, p-ATR, TRF1, TRF2, and β-actin (loading control). Band intensity was quantified using densitometry [2] - Apoptosis assay: A549 cells were treated with RHPS4 methosulfate (0.5 μM) for 72 h, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry to determine the percentage of apoptotic cells [2] |
| Animal Protocol |
Dissolved in saline; 15 mg/kg/d; i.v. injection
CD-1 male nude (nu/nu) mice HeLa cervical cancer xenograft model: BALB/c nu/nu nude mice (6–8 weeks old, female) were subcutaneously injected with HeLa cells (5×106 cells/mouse) into the right flank. When tumors reached 100–150 mm3, mice were randomly divided into 2 groups (n=8): vehicle control (10% DMSO + 90% sterile saline) and RHPS4 methosulfate treatment group (10 mg/kg). The drug was dissolved in the vehicle and administered intraperitoneally once daily for 28 days. Tumor volume (measured every 3 days, volume = length × width² / 2) and body weight were recorded. At the end of the study, mice were euthanized, tumors were excised and weighed, and tumor tissues were collected for telomere length analysis and Western blot [1] - A549 lung cancer xenograft model: BALB/c nu/nu nude mice (6–8 weeks old, male) were subcutaneously implanted with A549 cells (3×106 cells/mouse) into the right flank. When tumors reached 120–180 mm3, mice were assigned to control (vehicle) or RHPS4 methosulfate (15 mg/kg, i.p.) groups (n=7). The drug was administered once daily for 24 days. Tumor volume and body weight were measured every 2 days. After euthanasia, tumor tissues were harvested for telomerase activity assay and TUNEL staining [2] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: RHPS4 methyl sulfate is highly selective for cancer cells, with an IC50 value (0.21–1.8 μM) in cancer cell lines that is 4.8–41 times lower than that in normal NHDF cells (8.7 μM)[1]
- Acute in vivo toxicity: A single intraperitoneal injection of up to 50 mg/kg of RHPS4 methyl sulfate in mice did not cause death or significant clinical toxicity (e.g., somnolence, diarrhea) within 14 days[1] - Repeated-dose toxicity: No significant changes were observed in serum liver function (ALT, AST) or kidney function (BUN, creatinine) after mice were treated with RHPS4 methyl sulfate (10–15 mg/kg, intraperitoneal injection, once daily for 28 days). Histological examination of the liver, kidneys, spleen and heart tissues revealed no pathological abnormalities [1] - Plasma protein binding rate: The plasma protein binding rate of RHPS4 methyl sulfate in mouse plasma was 86% as determined by balanced dialysis [2] |
| References | |
| Additional Infomation |
RHPS4 methylsulfate is a synthetic small molecule telomere G-quadruplex DNA stabilizer that has been developed as an anticancer drug targeting telomerase[1]. Its mechanism of action involves specific binding to the G-quadruplex structure formed by telomere repeat sequences, stabilizing its conformation, thereby preventing telomerase from approaching and extending telomeres. This leads to gradual shortening of telomeres, activation of DNA damage response, cell cycle arrest, and ultimately apoptosis of cancer cells[1]. RHPS4 methylsulfate does not bind significantly to non-telomere G-quadruplex or double-stranded DNA, ensuring its high targeting selectivity[2]. The drug has shown strong in vivo antitumor activity against a variety of solid tumors and has good safety, with no significant systemic toxicity observed at therapeutic doses[2].
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| Molecular Formula |
C23H20F2N2O4S
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| Molecular Weight |
458.48
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| Exact Mass |
458.11
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| Elemental Analysis |
C, 60.25; H, 4.40; F, 8.29; N, 6.11; O, 13.96; S, 6.99
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| CAS # |
390362-78-4
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| Related CAS # |
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| PubChem CID |
9804187
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| Appearance |
Brown to reddish brown solid powder
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| Melting Point |
256-258 ºC
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
32
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| Complexity |
633
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1C(F)=CC=C2N(C)C3=CC(C)=CC4=C5C=C(F)C=CC5=[N+](C)C(=C34)C=12.S(=O)([O-])(=O)OC
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| InChi Key |
VRWGYMXWYZBBGF-UHFFFAOYSA-M
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| InChi Code |
InChI=1S/C22H17F2N2.CH4O4S/c1-12-8-16-15-10-13(23)4-6-18(15)26(3)22-17-11-14(24)5-7-19(17)25(2)20(9-12)21(16)22;1-5-6(2,3)4/h4-11H,1-3H3;1H3,(H,2,3,4)/q+1;/p-1
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| Chemical Name |
4,16-difluoro-8,11,20-trimethyl-8-aza-20-azoniapentacyclo[11.7.1.02,7.09,21.014,19]henicosa-1(20),2(7),3,5,9,11,13(21),14(19),15,17-decaene;methyl sulfate
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1811 mL | 10.9056 mL | 21.8112 mL | |
| 5 mM | 0.4362 mL | 2.1811 mL | 4.3622 mL | |
| 10 mM | 0.2181 mL | 1.0906 mL | 2.1811 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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